1. Field of the Invention:
The present invention relates to regiospecific conjugate formation of oligosaccharide-containing antibodies by means of oxidative modification of the carbohydrate region of the antibodies.
2. Description of the Background:
The fixation of biologically active substances to substrates, for example, carrier materials etc., is one of the standard processes in biochemical research and engineering. Various techniques for this have been developed, which depend on the chemical nature of the substances to be fixed and the purpose of the fixation. See, e.g., Buchholz, K., "Characterization of Immobilized Biocatalysts", in Dechema Monographs, V. 84 No. 1724-1731, pub. Verlag Chemie (1979).
From the beginning, polysaccharides such as cellulose, starch, and dextran have been important in practice as carrier materials. Ordinarily these materials must be chemically modified for activation. A large number of modification methods have been proposed, notably the activation of starch by oxidation with periodate, wherein formyl groups are formed with cleavage of the hemiacetal ring. See Goldstein, L., Pecht, M., Blumberg, S., Atlas, D., and Levin, Y., Biochem., 9, 2322 (1979).
Coupling over the carbohydrate portion of cell membranes has been achieved, e.g., with red blood corpuscles by oxidation with periodate. The resulting reactive aldehyde groups couple with the side chain amino groups of proteins. See Sanderson, C.I., and Wilson, D.V., 1971, Immunochemistry, 8:163. Periodate oxidation of antibodies has also been used for conjugation of antibodies with enzyme markers. See Gaivoronskaya, A.G. et al., CA 95, 130787j. Similarly, in Eur. OS 0 175 617 conjugates were proposed which are formed from antibodies and therapeutic agents, which conjugates are directed against a target antigen.
U.S. Pat. No. 4,671,958 contains a method of covalent binding of "linker" groups to specific sites of antibody molecules which can be used against desired target antigens.
Eur. OS 0 088 695 describes a number of methods for covalent binding of soluble or insoluble conjugate partners, such as chemical substances, carriers, etc., to antibodies. The conjugates formed are interesting with regard to the affinity-separation and purification methods, as well as possible diagnostic and therapeutic applications. The binding may occur in particular, among others, via the carbohydrate part of the antibodies. E.g., for monoclonal immunoassays, IgM is oxidized with galactose oxidase and catalase and is then condensed with phenylhydrazine-glycyl-glycyl-arginyl-7-amino-4-methylcoumarin. See also Rodwell, J.D. et al., Proc. Nat. Acad. Sci. USA/Immunology, 83, 2632-36 (1986).
M.M Chua et al. Biochem. Biophys. Acta, 800, 3:291 (1984) also achieved binding of immunoglobulin to liposomal membranes by mild oxidation of carbohydrate groups by periodate or galactose oxidase in the constant region of the heavy chain of the immunoglobulin, to produce aldehyde functions which could then react with hydrazide groups fixed to the membrane surfaces of the liposomes.
D.J. O'Shannessy et al. also proposed specific conjugation of polyclonal and monoclonal antibodies via their carbohydrate regions. First, aldehyde groups are formed by mild oxidation, and these are converted with hydrazine derivatives of biotin, suitable fluorescing dyes or enzymes to produce stabile antibody conjugates having full immunological activity. Also, the possibility of fixing to hydrazine-modified solid substrates has also been proposed. The support used for the immune affinity chromatography is, e.g., (commercial) agarose-adipic acid hydrazide. See J. Appl. Biochem., 7, 347-355 (1985). In another publication, O'Shannessy and W.L. Hoffmann reported the use of weak acid catalyzed condensations of oxidatively formed aldehyde groups on the oligosaccharide side chains of glycoproteins with hydrazine-functionalized agarose. The latter had been produced by reacting commercial activated ester-group-containing agarose with hydrazine hydrate. See Biological Chem. Hoppe-Seyler, 368, 7:767-8 (1987).
Polymer-bound antibodies and antigens have received progressively wider use in detecting the presence of specific substances. See "Encyclopedia of Polymer Science and Technology", pub. J. Wiley, Vol. 2, pp. 55-59 (1985). Particularly noteworthy uses are the detection of special hormones, blood components, normal and abnormal cell types, and pathogens, and the detection of molecules whose presence indicates a malignant tumor. Further, monoclonal antibodies are also employed in therapy. For example, it is possible to bind lethal agents, such as toxic substances or radioisotopes, to cancer-specific antibodies, with the aim of having such complexes seek out and combine with the target cells, thereby eventually destroying the cells without permanently injuring the surrounding tissues.
However, antibodies have only limited stability with regard to pH and the ionic strength of the medium which surrounds them. Thus, chemical modification of antibodies to produce covalent fixing, e.g. to carrier materials, is almost invariably accompanied by problems. At a minimum it is necessary that such fixing cause no degradation of the combining affinity of the antibody to the antigen. For example, conventional methods of immobilizing antibodies on solid carriers by means of cyanogen bromide, N,N'-carbonyldiimidazole, periodate oxidation, or epoxide chemistry, often lead to binding via nucleophilic active groups such as, e.g., amino, thio, or hydroxy groups, near the antigen combining site, which can result in a significantly decreased combining capability. See Yim, K., Biol. Chem. Hoppe-Seyler, 386, 7:785 (1987). It has been suggested that the composition of the polymeric carrier is the decisive factor in determining its properties and suitability as a carrier. Buchholz, K., Ed., in "Characterization of immobilized biocatalysts", loc. cit., 64. Accordingly, an abundant selection of potential carrier materials exists in the literature, such as inorganic materials, modified natural products, or synthetic organic polymers. In Yim (loc. cit.), the binding of the antibodies occurs by covalent bonding between thiosemicarbazide and silica gel.
In view of the expanding role which carrier-bound antibodies play in biochemistry and medicine, there is a continued great demand for optimized carrier systems based on polymeric carriers, wherein the effectiveness of the antibodies bound to the carriers is reduced as little as possible, while maintaining the optimal stability, range of applicability and efficiency of the carrier system.